Nodal调控肝癌血管生成拟态分子机制及其在预测肝癌复发转移中的作用

Recurrence and metastasis of hepatocellular carcinoma (HCC) after operation is the choke point of further increasing the long-term efficacy of operation on HCC. Pilot studies of vasculogenic mimicry (VM) showed that VM may be the leading cause of recurrence and metastasis of HCC after operation.In our prophase research, we have found that VM was associated with recurrence and metastasis of HCC after operation, and was independent adverse prognostic factor for both overall survival and disease-free survival. It is now unclear how VM work in recurrence and metastasis of HCC after operation. Recent evidence achieved by us revealed that Nodal was highly expressed in HCC tissue and highly metastatic HCC cell line. Furthermore, a significant association was also foud between expression of Nodal and of VM. The result indicated that Nodal played important roles in regulating the ability of HCC cells to display VM and promoting recurrence and metastasis of HCC after operation.On the basis of precedent researches, the experiment firstly uses small interfering RNA (siRNA) and gene transfection to regulate the Nodal gene expression. Next, effect of Nodal on proliferation, apoptosis, invasion, adhesion, VM formation and some genes related to VM formation will be observed in vitro. Effect of Nodal on growth, metastasis and VM formation of HCC cells will be studied in orthotopic transplantation HCC models in vivo. The expression of Nodal, VM and some genes related to VM formation in HCC tissues will also be assayed by tissue microarray and immunohistochemistry. The experiment aims to provide theoretical and practical basis for clinical prediction of recurrence and metastasis of HCC after operation and targeting therapy, by exploring the relation between Nodal and VM formation of HCC, and feasibility of Nodal as an important predictor and diagnosis index of recurrence and metastasis of HCC after operation and a new target for the therapy of HCC.

肝癌术后复发转移是进一步提高肝癌远期疗效的瓶颈，研究表明血管生成拟态（VM）是肝癌发生复发转移的重要原因。我们前期研究显示VM与肝癌复发转移有关，是影响肝癌术后生存的独立危险因素，但其形成机制尚不清楚。课题组近期发现Nodal在肝癌组织及高转移肝癌细胞株中高表达，并与VM表达存在一定相关性，提示Nodal可能在肝癌VM形成和肝癌复发转移中发挥着重要作用。本项目拟在前期工作基础上，应用RNA干扰及基因转染技术调控肝癌细胞Nodal表达，体外观察其对增殖、凋亡、侵袭、迁移、VM形成、VM形成相关基因的影响，体内观察其对裸鼠人肝癌原位移植模型中肝癌生长、转移及VM形成的影响。进而采用组织芯片及免疫组化方法检测肝癌组织中Nodal、VM及VM形成相关基因表达，以研究其与肝癌VM形成的关系及可能分子机制，并探讨其作为肝癌复发转移预测诊断指标的可行性，从而为临床预测肝癌复发转移和靶向治疗提供理论依据。

根治性肝切除术后的转移和复发仍是影响病人长期生存的主要障碍，因此需要新的标志物去预测肝癌预后及侵袭性。血管生成拟态（VM）是近年来在肝癌等高度侵袭性的恶性肿瘤中发现的一种全新的肿瘤内血管生成模式，与肿瘤的侵袭、转移等生物学行为以及预后有关。Nodal 是TGF-β 超家族成员，胚胎形成的重要调节因子。近年来在肿瘤治疗领域受到了越来越广泛的关注。虽然有报道其在肝癌组织中表达，但其在肝癌中作用、与VM的关系及预后意义并不清楚。本项目在前期工作基础上，应用RNA干扰及基因转染技术调控肝癌细胞Nodal表达，体外观察其对增殖、凋亡、侵袭、迁移、VM形成的影响；采用免疫组化方法检测肝癌组织中Nodal及VM表达以研究Nodal在人肝癌组织中表达意义及与VM形成、肝癌复发转移的关系；采用基因转染技术研究Nodal调控VM形成的机制。此外，还在研究的基础上拓展了肝癌相关研究，如Cripto-1在肝癌中的表达及其临床意义研究；肝癌组织中periostin（PN）和Snail表达的关系及共表达的预后意义研究；索拉非尼抑制巨噬细胞介导的肝细胞癌上皮间质转变(EMT)等。结果显示Nodal在高转移细胞株MHCC97H 和 HCCLM3中表达, 表明Nodal表达与细胞的恶性潜能有关。干扰后肝癌细胞生长、凋亡无明显影响，但干扰后细胞侵袭、迁移能力降低，能形成VM的MHCC97H三维培养下不能形成VM，而不能形成VM的肝癌细胞系HepG2转染后能形成VM。人肝癌组织中Nodal的高表达与肝癌侵袭转移有关，Nodal表达与VM形成正相关。Nodal高表达是影响肝癌患者术后生存的独立危险因素。Twist1可能在Nodal介导的VM形成中扮演重要角色。Nodal/Cripto-1信号通路在肝癌的侵袭转移及VM形成过程中可能发挥的调控作用。Cripto-1在肝癌组织中的表达与肿瘤血管侵犯、肿瘤分化程度及转移情况相关。PN/Snail共表达肝癌侵袭性强，其可能是一个新的不良预后独立预测因子。表明索拉非尼通过HGF-Met通路抑制极化巨噬细胞介导的肝癌细胞EMT。表明miR-122/PCDH20/Akt/mTOR信号轴在调节人肝癌化疗反应中起着重要的作用。探索Nodal对肝癌VM的影响机制及其在预测肝癌复发转移中的作用，将可能为临床预测肝癌复发转移和靶向治疗提供理论依据，从而具有重要的临床意义。